This invention relates to a reagent for immunoassay and an analytical method using the same, more particularly to a reagent for an immunoassay which is utilized to quantitatively analyze a specific antigen or antibody, an analytical method in which the reagent is employed, and an improvement in an immunoassay for quantitatively analyzing a specific antigen or antibody in a sample.
In recent years, a variety of tumor markers have been found out, as researches on cancers make advances. Typical examples of such tumor markers include .alpha.-fetoprotein (AFP), carcinoembryonic antigen (CEA), basic fetoprotein (BFP) and pancreatic oncofetal antigen (POA). Concentrations of these tumor markers in normal human sera are very low (e.g., the concentration of AFP is 10 ng/ml or less). On the other hand, these concentrations of tumor patients are about 10 times as high as the normal human sera in most cases. At any rate, with regard to the quantitative analysis of the tumor markers, a very high detection sensitivity is required.
In order to satisfy this requirement, there has been developed radio immunoassay (RIA) in which an antigen or antibody labelled by a radioactive material is employed. However, the RIA method has been troublesome in points of handling and disposal treatment. For this reason, there has been suggested another immunoassay in which an antigen or antibody labelled with a variety of materials such as enzymes and fluorescent materials in place of the radioactive materials. However, this immunoassay technique also has the drawback that a free antibody and a combined antibody must be separated from each other in any manner. Further, an EMIT method, which has been disclosed in Rosenthal A. F., Vargas M. G. and Klass C. S. (1976), Clin. Chem., Vol. 22, p. 1899, is an epochal method by which measurement can be made in a uniform system without requiring any separation process, but it cannot be fundamentally applied to a protein antigen or antibody having a high molecular weight.
Haxby J. A., Kinsky C. B. and Kinsky S. C. (1968), Biochemistry, Vol. 61, p. 300 has revealed a method by which there is prepared liposomes where a fat-soluble antigen is taken in its membrane and glucose is contained, and glucose discharged from the liposomes due to its breakage caused by an antigen-antibody reaction is quantitatively determined in order to accomplish the quantitative analysis of an antibody. However, if the measurement of the tumor marker is attempted, it will be necessary to support the marker itself or the antibody against this marker, i.e. an immunogloblin which is a kind of protein, on the liposomes. Up to now, the liposomes incorporated into hydrophobic proteins have been feasible, but it has not been reported yet that water-soluble protein-bearing liposomes are contemplated for the aim of immunoassays of antigens or antibodies. This reason is that a technique of supporting the water-soluble protein on liposomes has not been established.
Further, in Japanese Provisional Patent Publication (KOKAI) No. 132564/1981 entitled "Product for immunological analysis and method for preparing the same", there is disclosed a method by which liposomes supporting an antigen or antibody thereon and including an enzyme therein are used to carry out an immunoassay. In this suggested method, a protein is supported on the liposomes by using a bifunctional cross-linking agent such as glutaraldehyde. However, it has been realized from the present inventors' researches that if the antigen is supported on the liposomes with the aid of the cross-linking agent, the activity of the antigen will be generally reduced and the liposomes breakage to be caused by the antigen-antibody reaction will not be achieved.
Furthermore, conventional immunoassay techniques have drawbacks of usually taking a long time and not being able to measure automatically a large amount of samples.
Used for the quantitative analysis of specific antigens or antibodies present in a sample is, for example, radio immunoassay (hereinafter "RIA"). Since, however, a radioactive element is employed in RIA, it has the problems that an equipment for its exclusive use must be installed, operation must be carried out by a qualified operator and, moreover, waste is required to be disposed with caution.
Known as another analytical method is, for example, immunoelectrophoresis. The immunoelectrophoresis, however, has the problems that it requires a long time for the assay, has a low sensitivity, and can not be applied when there is contained only a very trace amount of target substances (or substances to be detected).
Now, in an earlier Japanese Patent Application No. 224509/1983, the present inventors have disclosed a liposome reagent comprising a hydrophilic antibody or antigen immobilized on its surface and a hydrophilic marker material enclosed in its inside. The analytical method employing this reagent can be summarized as follows: The above liposome reagent is added to the sample in which antigens or antibodies are present and a complement is separately added thereto, so that liposomes are destroyed by the antigen-antibody reaction and by the action of the complement to be accompanied with it and the marker materials enclosed (for example, fluorescent chemical substances) are released. Since there is a correlation between the quantity of this marker materials released and the quantity of the target substances in the sample, the target substances can be determined by determining the released marker materials according to a given analytical method (for example, a fluorescent analysis). Employment of this reagent may cause no problems that may be caused in RIA, and thus the immunoassay can be expected to be simplified.
However, when a sample containing serum or protein is analyzed with use of this liposome reagent, it has come to be found that a nonspecific reaction takes place independent of the antigen-antibody reaction to cause the destruction of liposomes. This is presumed to be caused by the reaction between the protein or trace chemical substances and the liposomes. For this reason, it has been conventionally practiced that the sample such as a serum is diluted to carry out the analysis.
For example, when a reagent comprising an anti-human alpha-fetoprotein antibody (hereinafter "anti-human AFP antibody") immobilized on liposomes is used to analyze AFP in serum, it has been practice to dilute human serum to 1/100 in order to eliminate the influence by the nonspecific reaction. However, a normal human serum contains only 10.sup.-8 g/ml or less. Therefore, the dilution of normal human serum to 1/100 may result in the measurement corresponding to an AFP concentration of 10.sup.-10 g/ml or less (for example, the fluorescent analysis), bringing about the problem that a precise determination can be hardly carried out.